Microarray preparation methods for synthetic oligomers, including oligonucleotides (oligos) include the following: (1) spotting a solution on a prepared flat or substantially planar surface using spotting robots; (2) in situ synthesis by printing reagents via ink jet or other computer printing technology and using standard phosphoramidite chemistry; (3) in situ parallel synthesis using electrochemically generated acid for removal of protecting groups and using standard phosphoramidite chemistry; (4) in situ synthesis using maskless photo-generated acid for removal of protecting groups and using regular phosphoramidite chemistry; (5) mask-directed in situ parallel synthesis using photo-cleavage of photolabile protecting groups (PLPG) and standard phosphoramidite chemistry; (6) maskless in situ parallel synthesis using PLPG and digital photolithography and standard phosphoramidite chemistry; and (7) electric field attraction/repulsion for depositing fully formed oligos onto known locations.
An electrode microarray for in situ oligo synthesis using electrochemical deblocking is disclosed in Montgomery U.S. Pat. Nos. 6,093,302; 6,280,595, and 6,444,111 (Montgomery I, II, and III respectively), all of which are incorporated by reference herein. Another and materially different electrode array (not a microarray) for in situ oligo synthesis on surfaces separate and apart from electrodes using electrochemical deblocking is disclosed in Southern U.S. Pat. No. 5,667,667, which is incorporated by reference herein. Photolithographic techniques for in situ oligo synthesis are disclosed in Fodor et al. U.S. Pat. No. 5,445,934 and the additional patents claiming priority thereto, all of which are incorporated by reference herein. Electric field attraction/repulsion microarrays are disclosed in Hollis et al. U.S. Pat. No. 5,653,939 and Heller et al. U.S. Pat. No. 5,929,208, both of which are incorporated by reference herein. A review of oligo microarray synthesis is provided by: Gao et al., Biopolymers 2004, 73:579.
For microarrays, a photon-based detection system (i.e., optical detection) is generally used to detect a binding event. Most commonly, microarray detection processes use fluorescent tags on the targets for transduction of a binding event on a microarray. Chemiluminescent systems are also used. The amount of binding is related to the amount of fluorescence measured. Alternatively, visible dyes or luminescent tags may be used. For example, for DNA hybridization, the tag is attached to target DNA sequences to detect hybridization to a probe oligonucleotide attached to a microarray. Depending upon the intensity of the signal from the tag, such microarrays may have to be read through laser confocal microscope-based system for microarrays configured in a monolayer (such as those microarrays made through high density spotting or photolithography techniques) or by a video-type camera (such as a CCD camera) for those microarrays having a three-dimensional matrix for each spot in high density formats.
An alternative to fluorescence has been optical detection of probe-target binding. In a so-called scanometric assay, targets are labeled with catalytic gold nanoparticles. After binding with the probe, a silver salt is added to the solution and metallic silver is deposited where the nanoparticles are bound. Detection is similar to optical photographic development and is recorded using either a digital scanner or photographic techniques. This technique does alleviate some of the technical demands of fluorescent detection but it is unclear how sensitive scanometric techniques will be at spot sizes relegated by current state of the art microarrays.
Generally, photon-based readers are expensive, relatively large and cumbersome, extremely heavy and unsuitable for field-based deployment, rely on sophisticated numerical algorithms, and must be accurately calibrated before use; thus, use of such readers is generally limited to a laboratory setting. In each instance of “reading” the signal from a microarray, there is often stray light or other noise signals that cause false or inaccurate readings. Moreover, distinguishing between shades of gray or barely perceptible signals as true positives or false positives is difficult. Finally, there may be quenching of the fluorescent signal and auto absorption of the signal by other labels within close proximity to the bound target. The additional complexity associated with using a photon-based reader imparts added variability. Therefore, there is a need in the art for improvements to the detection process for analyzing binding events on microarrays. The present invention was made to address this need to improve detection of binding events on an electrode microarray by basing detection on electrical properties rather than light properties of electrodes having binding events.